Treatment of cells with type I interferon (IFN) leads to transcriptional activation of genes involved in cellular antiviral effects, growth inhibition, and immune regulation. The study of IFN-mediated transcription is critical to treatment of chronic viral diseases including HIV, the causative agent of AIDS and Hepatitis C, the leading cause of liver failure and cirrhosis. Both viruses are endemic in the United States and IFN has been used in their treatment. The long term objective of my laboratory is to understand the mechanisms by which STAT and IRF transcription factors combine to produce the trimeric IFN-responsive transcription complex, ISGF3. The ISGF3 factor is a unique STAT-dependent transcription complex because the signaling phase is dissociated from the nuclear functions by an obligatory DNA binding subunit, IRF9/p48. Although the signal transduction pathways involved in activation of the ISGF3 trimeric transcription factor complex have been well studied, the mechanism of transcriptional activation is only poorly understood. The published literature and our own preliminary studies indicate that a fundamental transcriptional activation domain (TAD) for ISGF3 resides in the STAT2 C-terminus. Preliminary studies using IRF9-STAT2 TAD fusion proteins that mimic ISGF3 transcriptional responses and recapitulate IFN biological responses have produced a structure-function map of this domain and have contributed to generating the hypothesis that the STAT2 TAD provides a protein interaction platform for several types of transcriptional co-activators. In support of this hypothesis, we have used a candidate-partner approach to identify a new transcriptional partner for STAT2 that is a subunit of the metazoan Mediator complex. Analysis of endogenous ISGF3 activity in STAT2 mutant-expressing cell lines will be carried out to connect specific STAT2 TAD regions with specific target gene expression patterns, biological responses, and promoter chromatin remodeling activities. The four proposed experimental aims will: (1) determine the functional significance of transcriptionally important STAT2 C-terminal TAD regions in a physiological context, (2) characterize the molecular mechanisms involved in STAT2 interactions with Mediator subunits, (3) determine the ability of ISGF3 to regulate promoter chromatin dynamics and map corresponding STAT2 C-terminal regions, and (4) identify additional protein partners for the STAT2 C-terminus. The proposed experiments provide a unique model system for the study of activated transcription, in which contributions of specific STAT2 TAD regions responsible for IFN target gene expression patterns, co-activator recruitment, and/or chromatin remodeling activities can be correlated with physiologically relevant IFN biological responses.